Sypro orange fluorescence dye

Ligand binding usually stabilizes the protein during the thermal denaturation process, and a tighter binder causes a larger positive shift in the protein melting temperature (T_m) (38 (link)). The binding of ligands to SaMetRS was evaluated by the fluorescence-based thermal shift assay as described (39 (link)). Briefly, mixtures with a final volume of 20 μl that contained 4 μg of SaMetRS-FL, 4 × SYPRO orange fluorescence dye (Sigma-Aldrich) and different ligands (10 μM REP8839, 5 mM L-Met, 5 mM ATP, or 1 mM fragments) in TSA buffer (100 mM Tris pH 8.0, 150 mM NaCl) were prepared in 96-well plates. The mixtures were incubated at 25°C for 10 min and then heated from 25°C to 95°C at a rate of 1°C/min. The fluorescence intensity was recorded every 20 s by using a StepOnePlus Real-Time PCR instrument (Life Technologies). The melting curves, using the fluorescence signal as the ordinate and the temperature as the abscissa, were fitted by using StepOne™ software v2.3 to obtain the melting temperature (T_m) of the protein. Triplicate assays were performed, and the average T_m values were used. The thermal stabilization of SaMetRS that was caused by a ligand (a fragment, substrate or inhibitor) was termed ΔT_m, and it was calculated by subtracting the T_m of SaMetRS without the ligand from the T_m of SaMetRS supplemented with the ligand as follows:

Ligand binding usually stabilizes a protein during its thermal denaturation process, resulting in a positive shift of the protein melting temperature compared to the apo protein (34 (link)). The fluorescence-based thermal shift assay (TSA) was employed for fragment screening against asymmetric EcTrpRS bound with a molecule of TrpAMP. The 20 μl reactions, consisting of 150 mM NaCl, 100 mM MES pH 6.5, 2 μg EcTrpRS, 4 × SYPRO orange fluorescence dye (Sigma-Aldrich) and 1 mM of one of the tested fragments, were prepared in the 96-well plates (Life Technologies) on ice. The reaction without adding any fragment was used as the blank control. The plates were incubated at 25°C for 10 min and then heated from 25°C to 95°C at a rate of 1°C/min. The fluorescence intensity was recorded every 30 s using a StepOne Plus™ RT-PCR equipment. The melting temperatures (T_m) of EcTrpRS with and without fragments were calculated using StepOne™ software v2.3. The average T_m values of triplicate assays were used. A fragment was considered as a positive hit when the ΔT_m between EcTrpRS with and without adding this fragment was greater than 1°C. Furthermore, a parallel fragment screening against the ‘open-open’ EcTrpRS (prepared by dialysis of the ‘open-closed’ asymmetric EcTrpRS) was performed as a control.

The binding of RM-A to IleRS and the impact of intermediate analogue (Ile-AMS, MedChemExpress) or substrates on binding were evaluated by the fluorescence-based thermal shift assay (TSA). Ligand binding usually stabilizes the protein during the thermal denaturation process, and tighter binding usually causes a larger positive shift in the protein melting temperature (T_m)^{45 (link)}. Briefly, mixtures with a final volume of 20 μL containing 150 mM NaCl, 100 mM HEPES (pH 7.5), 10% glycerol, 5 mM DTT, 2 μg of IleRS, 4× SYPRO orange fluorescence dye (Sigma-Aldrich) and different ligands (50 μM RM-A, 2 mM l-isoleucine, 2 mM ATP, or 50 μM Ile-AMS) were prepared in 96-well plates. The mixtures were incubated at 25 °C for 10 min and then heated from 25 °C to 95 °C at a rate of 1 °C/min. The fluorescence intensity was recorded by a StepOnePlus Real-Time PCR instrument (Life Technologies), and the maximums of the first derivatives were determined to calculate T_m values. Each T_m value was an average of triple assays.